Previous studies from our laboratory and others' have established that bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation, are augmented in response to certain cytokines and/or tissue ischemia, and home to sites of neovascularization. More recently, EPCs have been investigated as therapeutic agents to successfully enhance neovascularization. Just as classical angiogenesis may be impaired in certain pathologic phenotypes, however, ageing, diabetes, hypercholesterolemia, and hyperhomocysteinemia may likewise impair EPC function, including mobilization from the bone marrow and/or incorporation into neovascular foci. Gene transfer of EPCs during ex vivo expansion constitutes a potential means of addressing such putative liabilities in EPC function. Moreover, phenotypic modulation of EPCs during ex vivo expansion constitutes a potential means of addressing such putative liabilities in EPC function. Moreover, phenotypic modulation of EPCs ex vivo may also reduce the number of EPCs required for optimal transplantation post- ex vivo expansion, and thus serve to address a practical limitation of EPC transplantation post-ex vivo expansion, and thus serve to address a practical limitation of EPC transplantation, namely the volume of blood required to extract an optimal number of EPCs for autologous transplantation. The experiments outlined in this Proposal will investigate the thesis that gene transfer can be employed to achieve phenotypic modulation of EPCs. The Proposal has three specific aims. First, we will perform in vitro assays, as well as in vivo experiments in animal models of tissue ischemia, to optimize the methodology and effectiveness for EPC gene transfer of angiogenic growth factors. Second, we will investigate the role of the serine-threonine protein kinase, Akt (also known as protein kinase beta), in the biological activation of EPCs, using gene transfer strategies involved constitutively active and dominant negative forms of Akt. Third, we will investigate the impact of telomerase reverse transcriptase (TERT) gene transfer on EPC function and survival, in vitro and in vivo. We anticipate that the results of these experiments will yield new insights regarding both the fundamental biology as well as therapeutic applications of EPCs for postnatal neovascularization. If we are successful, these results will resolve certain issues sufficiently to permit translation to clinical studies, and provide new questions that will stimulate additional inquiries.